Epithelial-mesenchymal transition (EMT) is a highly conserved cellular program that allows polarized, immotile epithelial cells to convert to motile mesenchymal cells. EMT is fundamental for tissue remodeling events during development, but this process is re-engaged in adults during wound healing, tissue regeneration, organ fibrosis, tumor invasion and metastasis. While the physiological and clinical significance of EMT is overwhelming, the precise molecular and functional features of EMT remain poorly characterized. Villin is an epithelial cell specific actin-binding protein that is expressed in mos significant amounts in the gastrointestinal, urogenital and respiratory tracts. Villin is a multifunctional protein that regulates epithelial cell plasticity and EMT amongst other functions. Studies done with the villin knockout mice have clearly demonstrated that the absence of villin impairs the ability of epithelial cells to respond to signals that regulate EMT, resulting in a deficiency in wound repair and cell migration. Despite these findings, how villin actuates changes in EMT remains to be determined. In our preliminary studies, we demonstrate for the first time that villin localizes to the nucleus and interacts directly with a transcriptional co-regulator, ZBRK1. Furthermore, we report that nuclear villin activates the expression of the transcriptional factor Slug to modulate epithelial plasticity and EMT. Most importantly, our studies suggest that mislocalization of villin away from the brush border membrane is prognostic of increased mortality in patients with colorectal cancer. Studies designed in this application wil test our novel hypothesis that cytoplasmic-nuclear trafficking of villin regulates the stability and/or turnover of the transcriptional co-regulator ZBRK1, thereby modulating the expression of the key transcriptional factor Slug to induce EMT. Additional studies are designed to link the abnormal nuclear localization of villin with metastasis in patients with colorectal cancer. Specifically, the goal of the proposed studies are: (i) to validate the molecular mechanism(s) that regulates the cytoplasmic-nuclear trafficking and nuclear retention of villin; (ii) to characterize the villin-ZBRK1 complex and its role in the expression of Slug, a key transcriptional activator of EMT and; (iii) to elucidate the function of nuclear villin in the regulation of EMT and metastasis. The experimental approach we have proposed combines mechanistic and functional biochemical, cell and molecular biological studies together with in vivo studies in the villin-/-, villin+/+, ApcMin/villin-/-; ApcMin/villin+/+, and severe combined immunodeficiency mice (SCID) mice to allow us to unravel the complex question of how EMT is regulated. Additional studies are proposed using two unique human colon cancer tissue microarray resources namely, a cohort of 334 clinical trial specimens from patients enrolled in phase III MAX trial and a tissue microarray (TMA) of 29 matched primary and metastatic colon tumor specimens. The long-term goal of our studies is to translate our findings into clinical outcomes to diagnose, prevent and/or treat fibrosis and metastasis.